The long-term goals of this work relate to regulation of gluconeogenesis by the hormones, glucagon, epinephrine and insulin in isolated hepatocytes. Using hepatocytes of the rat, rabbit and guinea pig, we propose to identify those gluconeogenic-glycolytic enzymes whose assayable activities can be altered by the hormones. Radioimmunoassay for the key enzymes will be developed so that activities can be correlated with concentrations. The fine structure of hepatocytes of all three species will be studied through scanning and transmission electron microscopy. Subsequently, immunoelectronmicroscopy, involving the use of ferritin or peroxidase, will be applied to identify the absolute and relative locations of three pair of non-equilibrium enzymes: (1) hexokinase and glucose-6-phosphatase; (2) phosphofructokinase and fructose diphosphatase, and (3) pyruvate kinase and phosphoenolpyruvate carboxykinase. Immunoelectronmicroscopy will also be used to identify the relative locations of "consecutive" enzymes in a gluconeogenic sequence to learn if the enzymes are constrained or randomized within their cytoplasmic locations. We believe that the nature of control is determined to a great extent by the absolute and relative locations of the enzymes. Since most of the enzymes of interest in gluconeogenesis are cytoplasmic, the proposed immunoelectronmicroscopy could give resolution to the cytoplasm beyond that obtained by traditional cell fractionations and electron microscopic techniques. It is further proposed to develop the use of antibodies as metabolic inhibitors against specific gluconeogenic-glycolytic enzymes in studies of carbohydrate metabolism. This is expected to provide information about the nature of control when control is dependent upon inhibition of some enzymes.